OBJECTIVE-Subjects with dietary obesity and pre-diabetes have an increased risk for developing both nerve conduction slowing and small sensory fiber neuropathy. Animal models of this type of neuropathy have not been described. This study evaluated neuropathic changes and their amenability to dietary and pharmacological interventions in mice fed a high-fat diet (HFD), a model of pre-diabetes and alimentary obesity.RESEARCH DESIGN AND METHODS-Female C57BL6/J mice were fed normal diets or HFDs for 16 weeks.RESULTS-HFD-fed mice developed obesity, increased plasma FFA and insulin concentrations, and impaired glucose tolerance. They also had motor and sensory nerve conduction deficits, tactile allodynia, and thermal hypoalgesia in the absence of intraepidermal nerve fiber loss or axonal atrophy. Despite the absence of overt hyperglycemia, the mice displayed augmented sorbitol pathway activity in the peripheral nerve, as well as 4-hydroxynonenal adduct nitrotyrosine and poly(ADP-ribose) accumulation and 12/15-lipoxygenase overexpression in peripheral nerve and dorsal root ganglion neurons. A 6-week feeding with normal chow after 16 weeks on HFD alleviated tactile allodynia and essentially corrected thermal hypoalgesia and sensory nerve conduction deficit without affecting motor nerve conduction slowing. Normal chow containing the aldose reductase inhibitor fidarestat (16 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) corrected all functional changes of HFD-induced neuropathy.CONCLUSIONS-Similar to human subjects with pre-diabetes and obesity, HFD-fed mice develop peripheral nerve functional, but not structural, abnormalities and, therefore, are a suitable model for evaluating dietary and pharmacological approaches to halt progression and reverse diabetic neuropathy at the earliest stage of the disease. Diabetes 56: [2598][2599][2600][2601][2602][2603][2604][2605][2606][2607][2608] 2007 O ver the last decade, profound changes in the quality, quantity, and source of food consumed in many developed countries combined with a decrease in levels of physical activity have led to an increase in the prevalence of diabetes and its complications (1). Furthermore, some manifestations of peripheral diabetic neuropathy (PDN) and cardiovascular disease in overweight and obese subjects develop at the stage of impaired glucose tolerance (IGT), preceding overt diabetes (2-4). A high BMI is a well-recognized risk factor for median nerve sensory conduction slowing and carpal tunnel syndrome (5-7). Furthermore, nondiabetic obese subjects have been reported to display significantly decreased compound muscle action potential amplitude of tibial and peroneal nerves and decreased sensory action potential amplitude of median, ulnar, and sural nerves compared with nondiabetic individuals (8). In the same study, warm and cold sensations from the index and little fingers, warm sensation from the big toe, and thermal and pain thresholds from the little finger directly correlated with the insulin sensitivity index, which was reduced in obese subjects. A higher prevalence...

Whereas functional, metabolic, neurotrophic, and morphological abnormalities of peripheral diabetic neuropathy (PDN) have been extensively explored in streptozotocininduced diabetic rats and mice (models of type 1 diabetes), insufficient information is available on manifestations and pathogenetic mechanisms of PDN in type 2 diabetic models. The latter could constitute a problem for clinical trial design because the vast majority of subjects with diabetes have type 2 (non-insulin dependent) diabetes. This study was aimed at characterization of PDN in leptin-deficient (ob/ob) mice, a model of type 2 diabetes with relatively mild hyperglycemia and obesity. ob/ob mice (ϳ11 weeks old) clearly developed manifest sciatic motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable (ϳ78%) loss of intraepidermal nerve fibers. They also had increased sorbitol pathway activity in the sciatic nerve and increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion (DRG). Aldose reductase inhibition with fidarestat (16 mg ⅐ kg ؊1 ⅐ d ؊1 ), administered to ob/ob mice for 6 weeks starting from 5 weeks of age, was associated with preservation of normal MNCV and SNCV and alleviation of thermal hypoalgesia and intraepidermal nerve fiber loss but not tactile allodynia. Sciatic nerve nitrotyrosine immunofluorescence and the number of poly(ADP-ribose)-positive nuclei in sciatic nerve, spinal cord, and DRGs of fidarestattreated ob/ob mice did not differ from those in nondiabetic controls. In conclusion, the leptin-deficient ob/ob mouse is a new animal model that develops both large motor and sensory fiber and small sensory fiber PDN and responds to pathogenetic treatment. The results support the role for increased aldose reductase activity in functional and structural changes of PDN in type 2 diabetes. Diabetes 55: 3335-3343, 2006 P eripheral diabetic neuropathy (PDN) is a devastating complication of diabetes and a leading cause of foot amputation (1,2). Clinical indications of PDN include increased vibration and thermal perception thresholds that progress to sensory loss, occurring in conjunction with degeneration of all fiber types in the peripheral nerve. A proportion of patients with PDN also describe abnormal sensations such as paresthesias, allodynia, hyperalgesia, and spontaneous pain that sometimes coexist with loss of normal sensory function (3). Functional, metabolic, neurotrophic, and morphological abnormalities of PDN have extensively been explored in animal models of type 1 diabetes and, in particular, in streptozotocin-induced diabetic rats (4 -8) and mice (9,10). In contrast, manifestations and pathogenetic mechanisms of PDN in type 2 diabetic models remain remarkably understudied despite the fact that the vast majority of subjects with diabetes have type 2 (non-insulin dependent) diabetes.The epidemic of obesity in the developed countries is driving a ...

A nonpsychoactive cannabinoid cannabidiol (CBD) has been shown to exert potent anti-inflammatory and antioxidant effects and has recently been reported to lower the incidence of diabetes in nonobese diabetic mice and to preserve the blood-retinal barrier in experimental diabetes. In this study we have investigated the effects of CBD on high glucose (HG)-induced, mitochondrial superoxide generation, NF-kappaB activation, nitrotyrosine formation, inducible nitric oxide synthase (iNOS) and adhesion molecules ICAM-1 and VCAM-1 expression, monocyte-endothelial adhesion, transendothelial migration of monocytes, and disruption of endothelial barrier function in human coronary artery endothelial cells (HCAECs). HG markedly increased mitochondrial superoxide generation (measured by flow cytometry using MitoSOX), NF-kappaB activation, nitrotyrosine formation, upregulation of iNOS and adhesion molecules ICAM-1 and VCAM-1, transendothelial migration of monocytes, and monocyte-endothelial adhesion in HCAECs. HG also decreased endothelial barrier function measured by increased permeability and diminished expression of vascular endothelial cadherin in HCAECs. Remarkably, all the above mentioned effects of HG were attenuated by CBD pretreatment. Since a disruption of the endothelial function and integrity by HG is a crucial early event underlying the development of various diabetic complications, our results suggest that CBD, which has recently been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in humans, may have significant therapeutic benefits against diabetic complications and atherosclerosis.

Poly(ADP-ribose) polymerase (PARP) activation, an important factor in the pathogenesis of diabetes complications, is considered a downstream effector of oxidativenitrosative stress. However, some recent findings suggest that it is not necessarily the case and that PARP activation may precede and contribute to free radical and oxidantinduced injury. This study evaluated the effect of PARP inhibition on oxidative-nitrosative stress in diabetic peripheral nerve, vasa nervorum, aorta, and high glucoseexposed human Schwann cells. In vivo experiments were performed in control rats and streptozocin (STZ)-induced diabetic rats treated with and without the PARP inhibitor 3-aminobenzamide (ABA) (30 mg ⅐ kg ؊1 ⅐ day ؊1 i.p. for 2 weeks after 2 weeks of untreated diabetes). Human Schwann cells (HSC) (passages 7-10; ScienCell Research Labs) were cultured in 5.5 or 30 mmol/l glucose with and without 5 mmol/l ABA. Diabetes-induced increase in peripheral nerve nitrotyrosine immunoreactivity, epineurial vessel superoxide and nitrotyrosine immunoreactivities, and aortic superoxide production was reduced by ABA. PARP-1 (Western blot analysis) was abundantly expressed in HSC, and its expression was not affected by high glucose or ABA treatment. High-glucose-induced superoxide production and overexpression of nitrosylated and poly(ADPribosyl)ated protein, chemically reduced amino acid-(4)-hydroxynonenal adducts, and inducible nitric oxide synthase were decreased by ABA. We concluded that PARP activation contributes to superoxide anion radical and peroxynitrite formation in peripheral nerve, vasa nervorum, and aorta of STZ-induced diabetic rats and highglucose-exposed HSC. The relations between oxidativenitrosative stress and PARP activation in diabetes are birather than unidirectional, and PARP activation cannot only result from but also lead to free radical and oxidant generation. Diabetes 54:3435-3441, 2005 O xidative-nitrosative stress produced by free radicals and oxidants contributes to nerve conduction deficits (1-3), metabolic changes (3,4), impaired neurotrophic support (5), neurovascular dysfunction (1,2), abnormal sensation, and pain (6,7), as well as morphological abnormalities (8) characteristic for peripheral diabetic neuropathy (PDN). Enhanced oxidative-nitrosative stress is manifest in peripheral nerve, dorsal root and sympathetic ganglia, and vasculature of the peripheral nervous system of animals with both type 1 and type 2 diabetes (1,3,9 -13). One of the important, currently considered as downstream, effectors of oxidative-nitrosative injury and associated DNA single-strand breakage is activation of the nuclear enzyme poly(ADPribose) polymerase (PARP). Once activated, PARP cleaves nicotinamide adenine dinucleotide (NAD ϩ ) with formation of nicotinamide and ADP-ribose residues, which are attached to nuclear proteins with formation of poly(ADPribosyl)ated protein polymers. The process leads to 1) NAD ϩ depletion and energy failure (16,17), 2) changes of transcriptional regulation and gene expression (18), and 3) po...

Poly(ADP-ribose) polymerase (PARP) activation is emerging as a fundamental mechanism in the pathogenesis of diabetes complications including diabetic neuropathy. This study evaluated the role of PARP in diabetic sensory neuropathy. The experiments were performed in control and streptozotocin-induced diabetic rats treated with or without the PARP inhibitor 1,5-isoquinolinediol (ISO; 3 mg ⅐ kg ؊1 ⅐ day ؊1 i.p.) for 2 weeks after 2 weeks without treatment. Diabetic rats developed thermal hyperalgesia (assessed by paw-withdrawal and tail-flick tests), mechanical hyperalgesia (von Frey anesthesiometer/rigid filaments and Randall-Sellito tests), tactile allodynia (flexible von Frey filaments), and increased flinching behavior in phases 1 and 2 of the 2% formalin pain test. They also had clearly manifest increase in nitrotyrosine and poly(ADPribose) immunoreactivities in the sciatic nerve and increased superoxide formation (hydroxyethidine method) and nitrotyrosine immunoreactivity in vasa nervorum. ISO treatment alleviated abnormal sensory responses, including thermal and mechanical hyperalgesia and tactile allodynia as well as exaggerated formalin flinching behavior in diabetic rats, without affecting the aforementioned variables in the control group. Poly(ADP-ribose) and, to a lesser extent, nitrotyrosine abundance in sciatic nerve, as well as superoxide and nitrotyrosine formation in vasa nervorum, were markedly reduced by ISO therapy. Apoptosis in dorsal root ganglion neurons (transferase-mediated dUTP nick-end labeling assay) was not detected in any of the groups. In conclusion, PARP activation contributes to early diabetic sensory neuropathy by mechanisms that may include oxidative stress but not neuronal apoptosis.

Evidence that poly(ADP-ribose) polymerase (PARP) activation plays an important role in diabetic complications is emerging. This study evaluated the role of PARP in rat and mouse models of advanced diabetic neuropathy. The orally active PARP inhibitor 10-(4-methylpiperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15427; formulated as a mesilate salt, 30 mg kg(-1) day(-1) in the drinking water for 10 weeks after the first 2 weeks without treatment) at least partially prevented PARP activation in peripheral nerve and DRG neurons, as well as thermal hypoalgesia, mechanical hyperalgesia, tactile allodynia, exaggerated response to formalin, and, most importantly, intraepidermal nerve fiber degeneration in streptozotocin-diabetic rats. These findings are consistent with the lack of small sensory nerve fiber dysfunction in diabetic PARP -/- mice. Furthermore, whereas diabetic PARP +/+ mice displayed approximately 46% intraepidermal nerve fiber loss, diabetic PARP -/- mice retained completely normal intraepidermal nerve fiber density. In conclusion, PARP activation is an important contributor to intraepidermal nerve fiber degeneration and functional changes associated with advanced Type 1 diabetic neuropathy. The results support a rationale for the development of potent and low-toxicity PARP inhibitors and PARP inhibitor-containing combination therapies.

Obrosova IG, Drel VR, Oltman CL, Mashtalir N, Tibrewala J, Groves JT, Yorek MA. Role of nitrosative stress in early neuropathy and vascular dysfunction in streptozotocin-diabetic rats. Am J Physiol Endocrinol Metab 293: E1645-E1655, 2007. First published October 2, 2007; doi:10.1152/ajpendo.00479.2007.-Evidence for important roles of the highly reactive oxidant peroxynitrite in diabetic complications is emerging. We evaluated the role of peroxynitrite in early peripheral neuropathy and vascular dysfunction in STZ-diabetic rats. In the first dose-finding study, control and STZ-diabetic rats were maintained with or without the potent peroxynitrite decomposition catalyst Fe(III)tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin (FP15) at 3, 5, or 10 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 in the drinking water for 4 wk after an initial 2 wk without treatment for assessment of early neuropathy. In the second study with similar experimental design, control and STZ-diabetic rats were maintained with or without FP15, 5 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 , for vascular studies. Rats with 6-wk duration of diabetes developed motor and sensory nerve conduction velocity deficits, mechanical hyperalgesia, and tactile allodynia in the absence of small sensory nerve fiber degeneration. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve and dorsal root ganglia. All these variables were dose-dependently corrected by FP15, with minimal differences between the 5 and 10 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 doses. FP15, 5 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 , also corrected endoneurial nutritive blood flow and nitrotyrosine, but not superoxide, fluorescence in aorta and epineurial arterioles. Diabetes-induced decreases in acetylcholine-mediated relaxation by epineurial arterioles and coronary and mesenteric arteries, as well as bradykinin-induced relaxation by coronary and mesenteric arteries, were alleviated by FP15 treatment. The findings reveal the important role of nitrosative stress in early neuropathy and vasculopathy and provide the rationale for further studies of peroxynitrite decomposition catalysts in long-term diabetic models.OXIDATIVE STRESS resulting from increased production of reactive oxygen species (ROS) and downregulation or insufficient upregulation of antioxidant defense is a well-recognized fundamental mechanism in diabetic complications. Superoxide is a primary free radical produced in diabetic and hyperglycemic conditions. Superoxide is rapidly converted to several other ROS, i.e., hydroxyl radicals (Fenton and Haber-Weiss reactions), peroxynitrite (reaction with nitric oxide), and hydrogen peroxide (reaction catalyzed by intrinsic superoxide dismutase) (22). The rate of superoxide reaction with nitric oxide exceeds by at least one order of magnitude the rates of other aforementioned reactions, which makes peroxynitrite the number one oxidant in biological systems (38,57,71). Peroxynitrite causes the following numerous cytotoxic effects (nitrosative stress): 1) protein nitration and nitrosylation, 2) DNA singl...

Whereas an important role of free radicals and oxidants in peripheral diabetic neuropathy is well established, the contribution of nitrosative stress and, in particular, of the highly reactive oxidant peroxynitrite, has not been properly explored. Our previous findings implicate peroxynitrite in diabetes-associated motor and sensory nerve conduction deficits and peripheral nerve energy deficiency and poly(ADP-ribose) polymerase activation associated with Type 1 diabetes. In this study the role of nitrosative stress in diabetic sensory neuropathy is evaluated. The peroxynitrite decomposition catalyst Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin (FP15) was administered to control and streptozotocin (STZ)-diabetic mice at the dose of 5 mg kg −1 day −1 (FP15), for 3 weeks after initial 3 weeks without treatment. Mice with 6-week duration of diabetes developed clearly manifest thermal hypoalgesia (paw withdrawal, tail-flick, and hot plate tests), mechanical hypoalgesia (tail pressure Randall-Sellito test), tactile allodynia (flexible von Frey filament test), and ~38% loss of intraepidermal nerve fibers. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, grey matter of spinal cord, and dorsal root ganglion neurons. FP15 treatment was associated with alleviation of thermal and mechanical hypoalgesia. Tactile response threshold tended to increase in response to peroxynitrite decomposition catalyst treatment, but still remained ~59% lower compared with non-diabetic controls. Intraepidermal nerve fiber density was 25% higher in FP15-treated than in untreated diabetic rats, but the difference between two groups did not achieve statistical significance (p=0.054). Nitrotyrosine and poly(ADP-ribose) immunofluorescence in sciatic nerve, spinal cord, and dorsal root ganglion neurons of peroxynitrite decomposition catalyst-treated diabetic mice were markedly reduced. In conclusion, nitrosative stress plays an important role in sensory neuropathy associated with Type 1 diabetes. The findings provide rationale for further studies of peroxynitrite decomposition catalysts in a long-term diabetic model. KeywordsIntraepidermal nerve fiber loss; Nitrosative stress; Peroxynitrite decomposition catalyst; Poly(ADPribose) polymerase; Tactile allodynia; Thermal hypoalgesia Evidence for important role of the potent oxidant peroxynitrite, a product of superoxide anion radicals with nitric oxide, in the pathogenesis of diabetes (Olcott et al., 2004;Szabo et al., 2002a;Pacher et al., 2007) and diabetic complications (Nangle et al., 2004;Obrosova et al., 2005b;Pacher et al., 2005;Pacher et al., 2007;Szabo et al., 2002a) is emerging. Accumulation of nitrotyrosine [NT, a footprint of peroxynitrite-and other reactive nitrogen species (RNS)-induced protein nitration] has been documented in vascular endothelium (Pacher et al., 2005;Szabo et al., 2002b), myocardium (Pacher et al., 2005), retina (Cheung et al., 2005;Obrosova et al., 2005c), and kidneys (Drel et al.,...